This invention relates to improved agents for the enzymatic determination of glucose.
The most important routine analysis in a clinical laboratory is blood sugar determination. This analysis is used for the detection and therapeutic control of diabetes mellitus and for the diagnosis of several other metabolic diseases.
Specific methods are required for the quantitative determination of blood sugar. The oldest methods of analysis are based on the reduction capacity of glucose, wherein substances such as potassium hexacyanoferrate III, picric acid, or copper(II) ions in an alkaline solution are employed as oxidizing agents. However, because this technique, when employed in connection with body fluids, detects all other reducing substances, e.g., creatine, creatinine, uric acid, ascorbic acid and glutathione, its lack of specificity no longer satisfies present requirements.
Another group of determination methods is based on the formation of furan derivatives from glucose, employing the water-liberating effect of concentrated acids, and the furans thereafter are reacted with aromatic amines, such as, for example, o-toluidine, to form dyes. Although this method is widely utilized as a routine analysis, the specificity thereof leaves much to be desired since, in addition to glucose, all other aldoses also participate in the reacion. A grave disturbance of this test occurs when determining glucose in the serum of patients who received infusions of dextran based plasma expanders, which causes turbidity in the test mixture. Also, the determination is rendered troublesome and time- consuming by the step of boiling with acid, which is required for condensation purposes.
In addition to this so-called o-toluidine method, two enzymatic processes are customarily employed for the determination of glucose, viz., the glucose oxidase method and the hexokinase method.
In the glucose oxidase method, the glucose is oxidized by oxygen to gluconic acid with glucose oxidase catalysis, thus producing hydrogen peroxide which, in turn, oxidizes a colorless chromogen to a dye in a catalyzed secondary reaction. As the catalyst in the secondary reaction, peroxidase can be utilized. A frequently employed chromogen is o-dianisidine. The glucose oxidase is specific for glucose but the secondary reaction is disturbed by substances which consume hydrogen peroxide. In body fluids, especially interfering are uric acid, ascorbic acid and catalase. Additional disadvantages are the long duration of the analysis and the fact that the chromogen o-dianisidine is quite toxic.
The glucose analysis having the highest specificity is generally considered to be the enzymatic determination with hexokinase/glucose 6-phosphate dehydrogenase. In this method, the glucose is phosphorylated with adenosine triphosphate/ hexokinase, and the thus-formed glucose 6-phosphate is dehydrogenated to gluconate 6-phosphate with glucose 6-phosphate dehydrogenase, wherein the hydrogen is transferred to nicotinamide adenine dinucleotide phosphate (NADP). The reduced coenzyme is then determined photometrically. Due to the central position of glucose 6-phosphate in the carbohydrate metabolism, this test is susceptible to disturbances by foreign enzymes, e.g., phosphoglucose isomerase, phosphoglucomutase and gluconate 6-phosphate dehydrogenase. Therefore, it is recommended to eliminate the interfering reactions by extrapolation. See H. U. Bergmeyer, "Methoden der enzymatischen Analyse" (Methods of Enzymatic Analysis), 2nd Ed. p. 1166, 1970, Chemie publishers, Weinheim. However, this makes the analytical method uncertain, cumbersome and time-consuming, e.g., about 20- 30 minutes for a single determination.
It has now been found that the disadvantages of the methods heretofore employed in practice for the determination of glucose can be avoided by employing the novel glucose dehydrogenase enzymatic agent of this invention.
It was known from the pertinent literature (Methods of Enzymology, vol. IX, 92-111, Acad. Press New York, London, 1966; R. P. Metzger et al., J. Biol. Chem. 238, 1769-1772 (1964), and 240, 2767-2771 (1965); and N. G. Brink, Acta Chem. Scand. 7, 1081-1088 [1953]) that glucose dehydrogenase has a number of unfavorable properties which argue against the use thereof as an analytical agent. For example, frequently the glucose dehydrogenases are bound to particles and/or prove to be not dependent on NAD or NADP, respectively, Furthermore, it is known that glucose dehydrogenase has a low activity and/or too low a stability. The Michaelis constants of glucose dehydrogenase to glucose are only 0.007 - 2, i.e., only in a 0.007-molar solution is the enzyme saturated with substrate to the extent of one-half. However, during the usual blood sugar analysis, only about 10.sup.-.sup.7 mole of glucose is available. Consequently, there has been a considerable prejudice in the literature against the use of glucose dehydrogenase for enzymatic glucose analysis.
By means of the present invention, it is possible for the first time to provide an agent and a process for the enzymatic determination of glucose employing glucose dehydrogenase which yields rapid and reliable results with easy handling.